Porous semiconductor-based optical interferometric sensor

ABSTRACT

The measurement of the wavelength shifts in the reflectometric interference spectra of a porous semiconductor substrate such as silicon, make possible the highly sensitive detection, identification and quantification of small analyte molecules. The sensor of the subject invention is effective in detecting multiple layers of biomolecular interactions, termed “cascade sensing”, including sensitive detection of small molecule recognition events that take place relatively far from the semiconductor surface.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This is a divisional of application Ser. No. 08/961,308, filedOct. 30, 1997, which is a continuation-in-part of application Ser. No.08/924,601, filed Sep. 5, 1997, whose disclosures are incorporatedherein by reference.

GOVERNMENTAL SUPPORT

[0002] This invention was made with governmental support under ContractNo. N00014-95-1-1293 by the ONR. The government has certain rights inthe invention.

TECHNICAL FIELD

[0003] This invention is related to solid state sensors and, moreparticularly, to the use and preparation of a porous semiconductor suchas a silicon wafer for the quantitative and qualitative analysis of ananalyte such as an organic analyte.

BACKGROUND OF THE INVENTION

[0004] Solid-state sensors and particularly biosensors have receivedconsiderable attention lately due to their increasing utility inchemical, biological, and pharmaceutical research as well as diseasediagnostics. In general, biosensors consist of two components: a highlyspecific recognition element and a transducing structure that convertsthe molecular recognition event into a quantifiable signal. Biosensorshave been developed to detect a variety of biomolecular complexesincluding oligonucleotide pairs, antibody-antigen, hormone-receptor,enzyme-substrate and lectin-glycoprotein interactions. Signaltransductions are generally accomplished with electrochemical,field-effect transistor, optical absorption, fluorescence orinterferometric devices.

[0005] It is known that the intensity of the visible photoluminescencechanges of a porous silicon film depend on the types of gases adsorbedto its surface. Based on this phenomenon, a simple and inexpensivechemical sensor device was developed and disclosed in U.S. Pat. No.5,338,415.

[0006] As disclosed in that patent, porous films of porous films ofsilicon (Si) can be fabricated that display well-resolved Fabry-Perotfringes in their optical reflectance properties. The production of aporous silicon (Si) layer that is optically uniform enough to exhibitthese properties may be important for the design of etalons (thin filmoptical interference devices for laser spectroscopy applications) andother optical components utilizing porous Si wafers. Suchinterference-based spectra are sensitive to gases or liquids adsorbed tothe inner surfaces of the porous Si layer.

[0007] Ever increasing attention is being paid to detection and analysisof low concentrations of analytes in various biologic and organicenvironments. Qualitative analysis of such analytes is generally limitedto the higher concentration levels, whereas quantitative analysisusually requires labeling with a radioisotope or fluorescent reagent.Such procedures are time consuming and inconvenient. Thus, it would beextremely beneficial to have a quick and simple means of qualitativelyand quantitatively detect analytes at low concentration levels. Theinvention described hereinafter provides one such means.

BRIEF SUMMARY OF THE INVENTION

[0008] The subject invention contemplates the detection and, if desired,measurement of the wavelength shifts in the reflectometric interferencespectra of a porous semiconductor substrate such as a silicon substratethat make possible the highly sensitive detection, identification andquantification of small molecules and particularly, small organicmolecules (i.e., carbon-containing molecules e.g., biotin, and thesteroid digoxigenin), short DNA oligonucleotides (e.g., 16-mers), andproteins (e.g., streptavidin and antibodies). The binding of inorganicspecies such as metal ions is also contemplated. Most notably, thesensor of the subject invention has been shown to be highly effective indetecting multiple layers of biomolecular interactions, termed “cascadesensing”, including sensitive detection of small molecule recognitionevents that take place relatively far from the silicon surface.

[0009] In an exemplary embodiment, a p-type silicon (Si) wafer(substrate) is galvanostatically etched in a hydrofluoric acid(HF)-containing solution. The etched wafer is rinsed with ethanol anddried under a stream of nitrogen gas. Reflection of white light off theporous silicon results in an interference pattern that is related to theeffective optical thickness. The binding of an analyte to a recognitionpartner immobilized in the porous silicon substrate results in a changein the refractive index, which is detected as a wavelength shift in thereflection interference pattern.

[0010] One benefit of the present invention is the provision of a devicefor detecting the presence of target (analyte) molecules such asbiological or organic compound molecules at very low concentrations.

[0011] An advantage of the present invention is the provision of a meansfor detecting the presence of multilayered molecular assemblies.

[0012] Still another benefit of the present invention is a device thatis capable of quantitatively detecting an analyte.

[0013] Still another advantage of the present invention is that thepresence of an analyte in a sample solution can often be detected byvisual inspection, and without the need for special apparatus.

[0014] Still further benefits and advantages will be apparent to aworker of ordinary skill from the disclosure that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] An understanding of the present invention will be facilitated byconsideration of the following detailed description of a preferredembodiment of the present invention, taken in conjunction with theaccompanying drawings, in which like reference numerals refer to likeparts and in which:

[0016]FIG. 1 is a schematic representation of the porous semiconductor,e.g., silicon-based optical interferometric biosensor of the presentinvention.

[0017]FIGS. 2A and 2B are interferometric reflectance spectra ofDNA-modified porous Si layers.

[0018]FIG. 3 shows the change in effective optical thickness in aDNA-A-modified porous Si layer as a function of DNA-A′ concentration.

[0019]FIGS. 4A, 4B, 4C and 4D are cascade sensing and reflectometricinterference spectra of multilayered molecular assemblies.

DETAILED DESCRIPTION OF THE INVENTION

[0020] A contemplated interferometric sensor is depicted in FIG. 1 andis extremely sensitive in detecting the presence of a number of ligands(analytes) that bind specifically to a chemical binder on the sensorsurface. For example, the lowest DNA concentration measured with acontemplated porous Si interferometric sensor was 9 fg/mm². Forcomparison, the detection limits of current technologies are:Interferometry (100 pg/mm²); Grating Couplers (2.5 pg/mm²); SurfacePlasmon Resonance (10 pg/mm²).

[0021] The devices and methods of the present invention employ a poroussemiconductor layer as an element of their interferometric sensors. A“porous semiconductor layer” is a porous layer having a relativelyconsistent thickness, relatively consistent porosity and made up of asemiconducting solid that is relatively transparent. A “semiconducting”material is one having a bulk resistivity of from about 1 to about 1×10⁷ohms per cm.

[0022] The term “transparent” as used herein refers to the property of amaterial to transmit a fraction, such as at least about 20% of asuitable range of wave lengths of light from which Fabry-Perot fringescan be generated.

[0023] The term “light” is employed herein to include not only thevisible portion of the electromagnetic spectrum, i.e. 350-800 nm, butalso the infrared region of from say 800-3000 nm and the ultravioletregion of from about 50-350 nm. Longer and shorter wavelengths can beemployed as well. The wavelengths employed can play a part in theselection of layer thickness and pore size of the porous semiconductorlayer. As a general rule shorter wavelengths permit thinner layerthicknesses and smaller pore sizes while longer wavelengths permitthicker layer thicknesses and larger pose sizes.

[0024] The porous semiconductor layer can range in thickness from about0.5 to about 30 microns with thicknesses of from about 1 or 2 to about10 microns being preferred when visible light such as white light isemployed and with thicknesses of from about 5 to about 30 microns beingpreferred with infrared wave lengths and thicknesses of from about 0.5to 5 microns being preferred with ultraviolet wave lengths.

[0025] The pores (or cavities) in the porous semiconductor layers aretypically sized in terms of their nominal “diameter” notwithstanding thefact that they are somewhat irregular in shape. These diameters rangefrom about 2 nm to about 2000 nm with diameters of from about 10 toabout 200 nm being preferred for visible light and 2-50 nm diametersbeing preferred for ultraviolet light and 100 to 2000 nm being preferredfor infrared light. The surface of the solid semiconductor is flat witha substantial degree of porosity such as from about 10% to about 80% ofthe surface area and typically from 20 to 70% of the surface area.

[0026] The semiconducting porous layer can be formed of anysemiconductor capable of being formed into the porous structure of thedesired thickness and porosity. Silicon and silicon alloys are preferredsemiconductors because of their amenability to the preferred galvanicetching process described herein for forming porous structures. Thesematerials can include p-doped silicon, n-doped silicon, intrinsic(undoped) silicon, as well as alloys of these materials with, forexample germanin in amounts of up to about 10% by weight as well asmixtures of these materials.

[0027] A representative device depicted in FIG. 1 is prepared from anelectrochemical etch of a semiconductor such as single-crystal p-type(boron-doped) silicon wafers that produce microporous silicon thatdisplays well-resolved Fabry-Perot fringes in its reflectometricinterference spectrum. Silicon-containing (silicious) semiconductors arepreferred herein, and although p-type silicon wafers are utilized hereinas exemplary substrates, it is to be understood that n-type silicon andundoped, intrinsic silicon can be used, as a silicon-germanium (Si—Ge)alloy containing up to about 10 mole percent germanium, Group IIIelement nitrides and other etchable semiconductor substrates. Exemplarysemiconductor substrates and dopants are noted below. n dopant p dopantH₂Se (CH₃)₂Zn H₂S (C₂H₅)₂Zn (CH₃)₃Sn (C₂H₅)₂Be (C₂H₅)₃Sn (CH₃)₂Cd SiH₄(ηC₂H₅)₂Mg Si₂H₆ B P Al As Ga Sb In

[0028] The substrate can be GaAs, Si, Al₂O₃, MgO, TiO₂, SiC, ZnO,LiGaO₂, LiAlO₂, MgAl₂O₄ or GaN.

[0029] Reflection of light at the top (surface) and bottom of theexemplary porous semiconductor layer results in an interference patternthat is related to the effective optical thickness (product of thicknessL and refractive index n) of the film by eq. 1,

mλ=2nL  (1)

[0030] where m is the spectral order and λ is the wavelength of light.Binding of an analyte to its corresponding recognition partner,immobilized on the porous silicon substrate area results in a change inrefractive index of the layer medium and is detected as a correspondingshift in the interference pattern.

[0031] The refractive index, n, for the porous semiconductor in use isrelated to the index of the semiconductor and the index of the materialspresent (contents) in the pores pursuant to eq. 2

n=(1−P)n _(semiconductor) +Pn _(contents)  (2)

[0032] Where P=porosity of porous semiconductor layer;n_(semiconductor)=refractive index of semiconductor;n_(contents)=refractive index of the contents of the pores.

[0033] The index of refraction of the contents of the pores changes whenthe concentration of analyte species in the pores changes. Mostcommonly, the analyte (target) species is an organic species that has arefractive index that is larger than that of the semiconductor. Thereplacement of a species of lower index of refraction (water) by anotherspecies of higher index of refraction (analyte) would be expected tolead to an increase in the overall value for index of refraction. Anincrease in index should result in a shift in the interference patternwavelengths to longer values; i.e., a bathochromic or “red” shiftpursuant to equation 1. Contrarily, the observed shift in interferencepattern wavelengths is opposite that which is expected; i.e., is towardshorter wavelengths exhibiting a hypsochromic or “blue” shift.

[0034] The basis for the observed wavelength blue shift is notunderstood with certainty. However, the observed, unexpectedhypsochromic shift in wavelengths is believed to be the result of areduction in the index of refraction of the semiconductor itself that isinduced by the intimate association of the semiconductor with the boundanalyte.

[0035] White light is preferred for carrying out reflectancemeasurements, and is used illustratively herein. The use of white lightor other light in the visual spectrum can permit a determination of thepresence of an analyte in a sample by visual inspection of a colorchange in the reflected light without the need of special apparatus. Itshould be understood, however, that reflected infrared (IR) andultraviolet (UV) light canals be utilized along with an appropriatespectral measuring device.

[0036] The sensors of the present invention include the binder molecule(also referred to as the “recognition partner”) for the analyte and thelike that is bound to or otherwise intimately associated with the poroussemiconductor surface. This intimate association can be accomplished byany approach that leads to the tethering of the binder molecule to thesemiconductor. This includes without limitation covalently bonding thebinder molecule to the semiconductor, ionically associating the bindermolecule to the substrate, adsorbing the binder molecule onto thesurface of the semiconductor, or the like. Such association can alsoinclude covalently attaching the binder molecule to another moiety,which in turn is covalently bonded to the semiconductor, bonding thetarget molecule via hybridization or another biological associationmechanism to another moiety with is coupled to the semiconductor.

[0037] The binding of an analyte to its corresponding recognitionpartner, immobilized on the porous silicon substrate, results in achange in refractive index of the layer medium and is detected as acorresponding shift in the interference pattern. Recognition partners orbinding compounds can be peptides, small molecules (molecular weight ofless than about 500), metal ions and their preferably organic bindingligands, antibodies, antigens, DNA, RNA or enzymes. More broadly, arecognition partner can be any receptor of an acceptor molecule that canbe adsorbed by the substrate and binds to a ligand provided by ofanother molecule or ion.

[0038] More specifically, the Examples that follow illustrate use of twodifferent single strands of binder DNA (SEQ ID NOs:1 and 2) bound to theporous silicon substrate, and two different single DNA strands (SEQ IDNOs:3 and 4, respectively) as analyte (Examples 1 and 3). Example 4illustrates the use of a biotin-bound porous silicon substrate withstrepavidin, as well as biotnylated anti-mouse antibodies that were usedto analyze for mouse-anti-digoxigenin, and those antibodies were thenused to assay for the presence of digoxigenin. Further exemplary bindingpairs include so-called polypeptide P-62 (SEQ ID NO:5) of U.S. Re.33,897 (1992), whose disclosures are incorporated by reference, withhuman antibodies to the Epstein-Barr nuclear antigen (EBNA) as analyte;monoclonal antibodies ATCC HB 8742 or HB 8746 that immunoreact withhuman apolipoprotein B-100 as analyte, or monoclonal antibodies ATCC HB9200 or HB 9201 that immunoreact with human apolipoprotein A-I asanalyte as are described in U.S. Pat. No. 4,828,986, whose disclosuresare incorporated by reference; and the several deposited monoclonalantibodies listed at column 13 of U.S. Pat. No. 5,281,710, and theirlisted binding partners as analyte, which disclosures are incorporatedby reference.

[0039] Electrochemical etching of Si can generate a thin (approximately1-10 μm) layer of porous Si on the silicon substrate with cavities ofabout 10 nm to about 200 nm in diameter, providing a large surface areafor biomolecular interaction inside the porous Si layer. The porousfilms are uniform and sufficiently transparent to display Fabry-Perotfringes in their optical reflection spectrum.

[0040] More particularly, a porous Si substrate is prepared by anelectrochemical etch of a polished (100)-oriented p-type silicon(B-doped 3 Ohm-cm resistivity) wafer. The etching solution is preparedby adding an equal volume of pure ethanol to an aqueous solution of HF(48% by weight). The etching cell is constructed of Teflon® and is opento air.

[0041] Si wafers are cut into squares with a diamond scribe and mountedin the bottom of the Teflon® cell with an O-ring seal, exposing 0.3 cm²of the Si surface. Electrical contact is made to the back side of the Siwafer with a strip of heavy aluminum foil, such as heavy duty householdaluminum foil. A loop of platinum wire is used as a counter-electrode.The exposed Si face can be illuminated with light from a tungsten lampfor the duration of the etch in order to enhance the optical propertiesof the films. Etching is illustratively carried out as a 2-electrodegalvanostatic operation at an anodic current density of 5 mA/cm² for 33minutes. After etching, the samples are rinsed in ethanol and driedunder a stream of N₂. Scanning electron microscopy and atomic forcemicroscopy showed that porous silicon films so prepared were about 5-10microns thick and contained an average of 200 nm diameter pores.

[0042] The porous semiconductor so prepared was modified by oxidationwith bromine gas in an evacuated chamber for one hour, followed byhydrolysis in air. The molecular recognition elements were then attachedto the resulting silicon dioxide surface using conventional techniques.

[0043] The sensors of this invention can be employed as discrete,independent units. Multiple sensors can also be arrayed together. Wheremultiple sensors are desired to be arrayed together, a plurality ofporous areas can be etched on to the surface of a single semiconductorsubstrate in much the same way as microchip patterns are prepared Aplurality of separate porous areas can also be combined to form adesired array.

[0044] An array of sensors can make it possible to have a plurality ofconcentrations of a single binder molecule on a single plate so as toprovide a “dose-response curve” for a particular analyte. Multiplesensors also can make it possible to have a plurality of differentbinder molecules on the same plate so as to make multiple screenings ina single test.

[0045] A sensor having a plurality of individual porous areas can beanalogized to a multi-well microtiter plate, and can contain the same ordifferent associated binder compound at any desired porous area so thatthe same or a different binding assay can be carried out on each porousarea. The individual binder compound-porous areas are then illuminated.Binding studies with analytes are then carried out for those areas,followed by reillumination. Binding results are obtained in a mannersimilar to that used for the individual porous areas exemplified herein.

[0046] Spectral Measurement. To measure optical interference spectra, aPrinceton Instruments CCD photodetector/Acton research 0.25 mmonochrometer, fitted with a fiber optic and microscope objective lensto permit detection from small (<1 mm²) sample areas was used for thestudies described here, but similar equipment is well-known and can beused instead. The white light source for the experiments was a lowintensity krypton, tungston or other incandescent bulb. A linearpolarizing filter was used to enhance the appearance of the interferencespectra.

[0047] The substrate can be pre-treated with a chemical receptor (bindercompound) species (such as an antibody) to provide chemical specificity.For gas measurements, the sample was mounted in a Pyrex® dosing chamberand exposed to the gaseous analyte of interest. For liquid-phasemeasurements, as in an aqueous medium, a Teflon® and O-ring cell similarto the cell employed in etching the porous layer was used. Measurementshave also been taken using a liquid flow-through chamber equipped withglass or plastic window.

[0048] The fringe pattern can be changed by replacing the air or liquidin the pores with a material of differing refractive index. The shift infringe maxima corresponds to a change in the average refractive index ofthe thin film medium. Solution of the simultaneous equations provided bymeasurement of the fringe spacing provides a quantitative measurementthat can be related to the analyte concentration. Chemical specificitycan be introduced by incorporating or chemically bonding molecularrecognition agents such as peptides, antibodies, antigens, single- ordouble-strand DNA or RNA, enzymes, a metal ion-binding ligand and thelike onto the inner surfaces of the porous Si film. Control measurementscan be performed on a similar sample that does not contain the molecularrecognition elements. Further details as to the preparation of a poroussilicon substrate and apparatus used for spectral measurements can befound in U.S. Pat. No. 5,338,415, whose disclosures are incorporated byreference.

[0049] Thus, one aspect of the invention contemplates a process fordetecting a analyte molecule such as an organic molecule analyte. Inaccordance with that process, a porous silicon substrate is provided andprepared, and that prepared substrate is provided and contacted with abinder compound to form a binder compound-bound substrate. Thewavelength maximum of the Fabry-Perot fringes is determined uponillumination of the binder compound-bound substrate. That bindercompound-bound substrate is thereafter contacted with a sample to beassayed that may contain an analyte that is an organic molecule thatbinds to the binder compound of the substrate. When the desired analyteis present in the sample, in distilled water or various buffer solutionsthat ligand binds to the binder compound to form a ligand-boundsubstrate. The contact between the sample and binder compound-boundsubstrate can be maintained for a few seconds to several hours, asdesired to form the ligand-bound substrate. When the substrate isthereafter reilluminated with the same light source, a shift in thewavelength maximum of the Fabry-Perot fringes from that previouslydetermined indicates the detection and therefore presence of the analytein the sample.

[0050] Without committing to any particular theory in support of thesubject invention, it is believed that the unique sensitivity of thesystem involves selective incorporation or concentration of an analytesuch as an illustrative organic analyte in the porous Si layer to modifythe refractive index by two effects: increase of the average refractiveindex of the medium in the pores by replacing water (refractive index1.33) with organic matter (refractive index typically 1.45), and alsodecrease of the refractive index of the Si by modifying the carrierconcentration in the semiconductor. A net increase in refractive indexis expected to shift the interference spectrum to longer wavelengths,whereas a decrease in index is expected to shift the spectrum to shorterwavelengths. Without exception, a shift to shorter wavelengths in suchcases has been observed, indicating that the induced change in thesemiconductor overwhelms the refractive index change occurring in thesolution phase.

[0051] Each of Examples 1-3 was carried out in 1.0 M aqueous NaCl at 25°C., whereas Examples 4 and 5 were carried out in 0.5 M NaCl.

EXAMPLE 1

[0052] Binder DNA oligonucleotide-derivatized porous silicon films wereemployed to test the selectivity and limits of detection of acontemplated sensor. For attachment of DNA, atrimethoxy-3-bromoacetamido-propylsilane linker was synthesized byreaction of bromoacetic acid with trimethoxy-(3-aminopropyl)silane inthe presence of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide-hydrochloride in methylenechloride as solvent The linker product was purified by columnchromatography on silica gel. The oxidized porous silicon samples werethen contacted with a toluene solution of the linker for 2 hours. Theresulting linker-bound substrate was thoroughly rinsed with pure tolueneand methylene chloride, and dried for about 18 hours under reducedpressure.

[0053] HPLC-Purified 5′-phopsphorothiate oligonucleotides (DNA-A andDNA-B, illustrated hereinafter) were separately dissolved at about 50nmol in a solution of 1:1:0.2; water/DMF/5% NaHCO₃ and admixed with thelinker-bound porous semiconductor substrate for about 2 hours. Thepresence of the DNA-modification on the porous surface of the resultingbinder compound-bound substrate was confirmed by FTIR spectroscopy.

[0054] In the presence of complementary analyte DNA sequences (DNAconcentrations ranging from 2×10⁻¹⁵ M to 2×10⁻⁶ M) pronounced wavelengthshifts in the interference pattern of the porous silicon films wereobserved (FIG. 2). Under similar conditions but in the presence ofnoncomplementary DNA sequences, no significant shift in the wavelengthof the interference fringe pattern was detected-only minor amplitudefluctuations were observed.

[0055] Specifically, measurements were made of two DNA sequences: DNA-A:5′-pGC CAG AAC CCA GTA GT-3′ SEQ ID NO:1 and DNA-B: 5′-CCG GAC AGA AGCAGA A-3′, SEQ ID NO:2

[0056] and corresponding complementary strands [(DNA-A′ (SEQ ID NO:3)and DNA-B′ (SEQ ID NO:4)]. For clarity, only one set of data are shown.

[0057] In FIG. 2A, the Fabry-Perot fringes 10 from a porous Si surfacederivatized with DNA-A are shown to shift to shorter wavelength 11 uponexposure to a 2×10⁻¹² M solution of DNA-A′ (the complementary sequenceof DNA-A) in 1 M NaCl (aq). The net change in effective opticalthickness (from 7,986 to 7,925 nm) upon DNA-A′ recognition isrepresented by the difference 12 between the two interference spectra.

EXAMPLE 2

[0058]FIG. 2B represents a control for Example 1, showing theFabry-Perot fringes 10 a of a DNA-A derivatized porous Si surface beforeand after exposure to a 2×10⁻¹² M solution of DNA-B (non-complementarysequence) in 1 M NaCl(aq). No wavelength shift was observed up to themeasured concentration of 10⁻⁹ M of DNA-B.

EXAMPLE 3

[0059] Fluorescence spectroscopy was used to independently investigatethe surface coverage of immobilized DNA on porous Si and the rate ofanalyte diffusion into the Si substrate for the purposes of comparisonwith the subject invention. Solutions of fluorescein-labeled analytecomplementary DNA oligonucleotides were placed in fluorescence cuvettesand the binder DNA-derivatized porous Si substrate was then added to thecell without stirring. At lowest DNA concentrations employed in thestudy, the fluorescence intensity of the samples decreased to anasymptotic limit in 40 min (similar equilibration times were observed inthe interferometric measurements described above) (FIG. 3). The dataindicate 1.1×10⁻¹² mol of bound DNA in a 1 mm² porous Si substrate(calculated from standardized fluorescence titration curves). The dataobtained from the reflectometric interference measurements also provideda similar coverage number.

EXAMPLE 4

[0060] The subject invention was used to sense multiple layers ofbiomolecular interactions (cascade sensing) and small moleculedetection. A linker with attached biotin was prepared by reaction ofIodoacetyl-LC-biotin (Pierce Biochemicals) with3-mercaptopropyl-trimethoxysilane (Aldrich Chemicals) indimethylformamide (DMF). After purification, the biotinylated linker wasdissolved in ethanol or DMF and the oxidized, porous semiconductor wasimmersed in the solution for 12 hours. The sample was then rinsedthoroughly with ethanol, and dried under a stream of nitrogen to providea binder compound-bound substrate.

[0061] Exposure of a biotinylated (binder) porous Si substrate to a5×10⁷ M analyte streptavidin solution resulted in a large blue-shift ofthe interference fringes, corresponding to a decrease in the measuredeffective optical thickness from 12,507-11,994 nm (the loweststreptavidin concentration employed was 10⁻¹⁴ M) (FIG. 4A). Controlstudies performed by exposing a biotinylated porous Si substrate toinactivated streptavidin (streptavidin pre-saturated with biotin) didnot display perceptible shifts in interference pattern.

[0062] The biotin-streptavidin monolayer surface was contacted withaqueous 10⁻⁸ M biotinylated anti-mouse IgG (from goat IgG). Binding ofthis secondary antibody to the surface was indicated by a decrease ineffective optical thickness of the monolayer from 11,997 to 11,767(lowest concentration employed with a detectable signal was 10⁻¹² M)(FIG. 4B). Treatment of the secondary antibody sample withanti-digoxigenin (mouse IgG) at a concentration of 10⁻⁸ M caused afurther decrease in the effective optical thickness of the monolayerfrom 11,706 to 11,525 nm (FIG. 4C). The interaction of digoxigenin (10⁻⁶M), a steroid with molecular weight of 392, with the anti-digoxigeninIgG-bound porous Si surface was also detected with a decrease of theeffective optical thickness from 11,508 to 11,346 nm (FIG. 4D).

EXAMPLE 5

[0063] To rule out the possibility of nonspecific interaction, anon-biotinylated surface was subjected to the same solution, andconditions as described in Example 4. No measurable change in theeffective optical thickness was observed on treatment with streptavidin,secondary antibody, primary antibody, and digoxigenin. Detection of therelatively small biotin molecule (MW=244) at concentrations as low as10⁻¹² M has also been demonstrated using biotin-streptavidin-modifiedporous Si.

[0064] Although the invention has been described with reference to apreferred embodiment, it will be understood by those skilled in the artthat various changes may be made and equivalents may be substituted forelements thereof without departing from the scope of the invention. Inaddition, many modifications may be made to adapt a particular situationor material to the teachings of the invention without departing from theessential scope thereof. Therefore, it is intended that the inventionnot be limited to the particular embodiment disclosed as the best modecontemplated for carrying out this invention, but that the inventionwill include all embodiments and equivalents falling within the scope ofthe appended claims.

[0065] Various features of the invention are set forth in the followingclaims.

1 5 1 16 DNA artificial sequence oligonucleotide 1 gccagaaccc agtagt 162 16 DNA artificial sequence oligonucleotide 2 ccggacagaa gcagaa 16 3 16DNA artificial sequence Complementary strand of seq. id no. 1 3actactgggt tctggc 16 4 16 DNA artificial sequence Complementary strandof seq. id. no. 2 4 ttctgcttct gtccgg 16 5 20 PRT artificial sequencesynthetic polypeptide related to Epstein-Barr virus nuclear antigen 5Ala Gly Ala Gly Gly Gly Ala Gly Gly Ala Gly Ala Gly Gly Gly Ala 1 5 1015 Gly Gly Ala Gly 20

What is claimed is:
 1. A process for detecting an analyte in a sample tobe assayed comprising the steps of: (a) providing a porous semiconductorsubstrate having a bound binder compound that forms a bindercompound-bound substrate and determining the wavelength of theFabry-Perot fringes upon illumination of said binder compound-boundsubstrate; (b) contacting said binder compound-bound substrate with asample to be assayed, said analyte present in said binding to saidbinder compound to form a ligand-bound substrate; and (c) thereafterreilluminating said substrate; whereby a shift in the wavelength maximumof the Fabry-Perot fringes indicates the detection of said analyte inthe sample.
 2. The process of claim 1 wherein said porous semiconductorsubstrate is silicon.
 3. The process of claim 1 wherein said bindercompound is an organic molecule.
 4. The process of claim 1 wherein saidanalyte is an organic molecule.
 5. A process for detecting an organicmolecule analyte in a sample to be assayed comprising the steps of: (a)providing a porous silicon semiconductor substrate having a bound bindercompound that forms a binder compound-bound substrate and determiningthe wavelength of the Fabry-Perot fringes upon illumination of saidbinder compound-bound substrate; (b) contacting said bindercompound-bound substrate with a sample to be assayed, said organicmolecule analyte present in said sample binding to said binder compoundto form a analyte-bound substrate; and (c) thereafter reilluminatingsaid substrate; whereby a shift in the wavelength maximum of theFabry-Perot fringes indicates the detection of said organic moleculeanalyte in the sample.
 6. The process of claim 5 wherein said providedsubstrate is prepared by the steps of: (a) etching said substrate; and(b) washing said etched substrate.
 7. The process of claim 5 whereinsaid binder compound is selected from the group consisting of peptides,antibodies, antigens, DNA, RNA, ligands that bind to metal ions andenzymes.
 8. The method of claim 5 wherein said contacting of step (b) iscarried out in an aqueous, liquid medium.
 9. A process of quantitativelydetecting organic analyte molecules in a sample comprising the steps of:(a) preparing a porous silicon semiconductor substrate; (b) contactingsaid substrate with a binder compound to form a binder compound-boundsubstrate and determining the wavelength of the Fabry-Perot fringes uponillumination of said binder compound-bound substrate; (c) introducing asample having an unknown concentration of an organic molecule analyte ata plurality of dilutions and measuring the shift in wavelength of theFabry-Perot fringes at said dilutions to prepare a first dose responsecurve of the unknown concentration of the organic molecule analyte; (d)providing a second, standard, dose response curve of Fabry-Perot fringewavelength shifts of known concentrations of the organic moleculeanalyte; and (e) comparing said first curve with said second curve on alog vs. log plot to thereby obtain the concentration of said organicmolecule analyte in said sample.
 10. In a solid state sensor fordetecting Fabry-Perot fringes from the reflection of light from asemiconductor substrate, the improvement comprising a semiconductorsubstrate having a porous surface, said substrate surface having anorganic binder compound adsorbed thereon.
 11. The sensor of claim 10further including a solution of an organic compound having an analytethat binds to said organic binder compound, said semiconductorsubstrate, when containing said analyte bound to said organic bindercompound reflecting light to exhibit Fabry-Perot fringe wavelengthsdifferent from those exhibited when the analyte is not so bound.
 12. Areflective sensor comprising a semiconductor substrate having a poroussurface area, said substrate surface area having an organic bindercompound intimately associated thereon, said binder compound bindingselectively with said analyte.
 13. The reflective sensor of claim 12wherein said semiconductor is a silicious semiconductor.
 14. The sensorof claim 13 wherein said silicious semiconductor is selected from thegroup consisting of intrinsic silicon, p-doped silicon, n-doped silicon,alloys of silicon and mixtures thereof.
 15. The sensor of claim 14wherein said alloys of silicon comprise silicon alloyed with up to about10% by weight of germanium.
 16. The reflective sensor of claim 12wherein said binder compound is selected from the group consisting ofpeptides, antibodies, antigens, DNA, RNA, ligands that bind to metalions and enzymes.
 17. The reflective sensor of claim 12 wherein saidsemiconductor surface defines a plurality of said porous surface areashaving an organic binder compound adsorbed thereon.
 18. A reflectivesensor for an analyte comprising a layer of porous semiconductor with abinder compound for the analyte intimately associated therewith, saidlayer being substantially transparent and having a top surface and abottom surface which reflect light to exhibit Fabry-Perot fringes havinga first set of characteristic wave lengths in the absence of analyte anda second set of characteristic wave lengths when analyte is present,said second set of characteristic wave lengths being detectably shiftedfrom said first set of characteristic wavelengths.
 19. The reflectivesensor of claim 18 wherein said layer of porous semiconductor with abinder compound for the analyte intimately associated therewith exhibitsa first index of refraction, wherein said analyte exhibits a secondindex of refraction which is greater than said first index ofrefraction, but wherein the second set of characteristic wavelengths isshorter than the first set of characteristic wave lengths.
 20. Thereflective sensor of claim 19 wherein said semiconductor comprisessilicon.
 21. An analytical sensor for detecting a target speciescomprising a porous semiconductor layer of a thickness selected togenerate Fabry-Perot fringes from the reflection of light therefrom,said Fabry-Perot fringes having a first set of characteristic peakwavelengths in the absence of the target species and a second set ofcharacteristic peak wavelengths in the presence of the target specieswith the second set of peak wave lengths being shifted toward shorterwavelengths relative to said first set of wavelengths.
 22. Theanalytical sensor of claim 21 wherein said semiconductor comprisessilicon.
 23. The analytical sensor of claim 22 wherein the poroussilicon has a first index of refraction and said target species has asecond index of refraction which is higher than said first index ofrefraction.
 24. The analytical sensor of claim 23 additionallycomprising a binder material intimately associated with the poroussilicon layer, said binder material specifically binding the targetspecies.
 25. A process for detecting a target species in a sample to beassayed comprising the steps of (a) selecting an assay sensor for thetarget species, the selected assay sensor comprising a layer of poroussemiconductor and a binder material intimately associated therewith,said binder material specifically binding the target species, said layerof a thickness selected to generate Fabry-Perot fringes from thereflection of light therefrom, said Fabry-Perot fringes having a firstset of peak wavelengths in the absence of the target species and asecond set of peak wavelengths in the presence of the target species;and (b) reflecting light off of the porous surface of the selected assaysensor in the presence of said sample and determining the presence orabsence of the target species in the sample from the Fabry-Perot fringesin the reflected light.
 26. The process of claim 25 wherein the poroussemiconductor comprises porous silicon.
 27. The process of claim 26wherein the target species is an organic target species.
 28. The processof claim 25 wherein said light comprises visible light.
 29. The processof claim 25 wherein said light is white light.
 30. The process of claim25 wherein said light comprises infrared light.
 31. The process of claim25 wherein said light comprises ultraviolet light.
 32. A reflectivesensor array for at least one analyte comprising a layer of poroussemiconductor with a plurality of discrete and separate regions havingone or more binder compounds for at least one of the at least oneanalytes intimately associated therewith, said layer being substantiallytransparent and having a top surface and a bottom surface which reflectlight in each of the plurality of regions to exhibit Fabry-Perot fringesfor such regions having a first set of characteristic wave lengths inthe absence of the at least one analyte and a second set ofcharacteristic wave lengths when analyte is present, said second set ofcharacteristic wave lengths being detectably shifted from said first setof characteristic wavelengths.